Wei Zhang, Thomas Dierk, Karle Christoph A, Kathöfer Sven, Schenkel Johannes, Kreye Volker A W, Ficker Eckhard, Wible Barbara A, Kiehn Johann
Department of Cardiology, Medical University Hospital Heidelberg, Heidelberg, Germany.
Chin Med J (Engl). 2002 May;115(5):668-76.
To investigate the molecular mechanism of human ether-a-go-go-related gene (HERG) potassium channels regulated by protein kinase A (PKA) in a human cell line.
HERG channels were stably expressed in human embryonic kidney (HEK) 293 cells, and currents were measured with the patch clamp technique. The direct phosphorylation of HERG channel proteins expressed heterologously in Xenopus laevis oocytes was examined by (32)P labeling and immunoprecipitation with an anti-HERG antibody.
Elevation of the intracellular cAMP-concentration by incubation with the adenylate cyclase activator, forskolin (10 micromol/L), and the broad range phosphodiesterase inhibitor, IBMX (100 micromol/L), caused a HERG tail current reduction of 83.2%. In addition, direct application of the membrane permeable cAMP analog, 8-Br-cAMP (500 micromol/L), reduced the tail current amplitude by 29.3%. Intracellular application of the catalytic subunit of protein kinase A (200 U/ml) led to a tail current decrease by 56.9% and shifted the activation curve by 15.4 mV towards more positive potentials. HERG WT proteins showed two phosphorylated bands, an upper band with a molecular mass of approximately 155 kDa and a lower band with a molecular mass of approximately 135 kDa, indicating that both the core- and the fully glycosylated forms of the protein were phosphorylated.
PKA-mediated phosphorylation of HERG channels causes current reduction in a human cell line. The coupling between the repolarizing cardiac HERG potassium current and the protein kinase A system could contribute to arrhythmogenesis under pathophysiological conditions.
在人细胞系中研究蛋白激酶A（PKA）调控人醚 - 去极化相关基因（HERG）钾通道的分子机制。
HERG通道在人胚肾（HEK）293细胞中稳定表达，采用膜片钳技术测量电流。通过（32）P标记和用抗HERG抗体进行免疫沉淀，检测非洲爪蟾卵母细胞中异源表达的HERG通道蛋白的直接磷酸化情况。
用腺苷酸环化酶激活剂福斯高林（10 μmol/L）和广谱磷酸二酯酶抑制剂异丁基甲基黄嘌呤（IBMX，100 μmol/L）孵育，使细胞内cAMP浓度升高，导致HERG尾电流降低83.2%。此外，直接应用膜通透性cAMP类似物8 - 溴 - cAMP（500 μmol/L）使尾电流幅度降低29.3%。细胞内应用蛋白激酶A的催化亚基（200 U/ml）导致尾电流降低56.9%，并使激活曲线向更正电位方向移动15.4 mV。HERG野生型蛋白显示出两条磷酸化条带，一条分子量约为155 kDa的上带和一条分子量约为135 kDa的下带，表明该蛋白的核心糖基化形式和完全糖基化形式均被磷酸化。
PKA介导的HERG通道磷酸化导致人细胞系中的电流降低。复极化心脏HERG钾电流与蛋白激酶A系统之间的偶联可能在病理生理条件下促成心律失常的发生。
